Systems for delivery and release of angiotensin-(1-7)

ABSTRACT

The prior art lacks a formulation, application or product of D-Ala7-Angiotensin-(1-7) (A- 779 ) and analogues and derivatives, D-Pro7-Angiotensin-(1-7) and analogues or derivatives or of Ang-(1-7) analogues or derivatives using cyclodextrins, liposomes, biodegradable polymers and its derivatives for the study or treatment of arterial hypertension and other cardiovascular diseases, wounds, burns, arrhythmia, tumors, diabetes mellitus, sperm mobility, nephropathy, gastrointestinal and gynecological disorders, angiogenesis, angioplatsy, alopecia and blood diseases in warm blooded animals, or as ligands for de G-protein-coupled receptor MAS. This characterizes the present invention as a more effective option for the study and treatment of pathologies associated or not to this receptor. A combination of two different technologies are provided: the molecular encapsulation of the peptide angiotensin-(1-7) and its analogues and derivates in cyclodextrin and the microencapsulation in biodegradable polymers and liposomes. It is also characterized by the increase of this peptides and its analogues and derivatives using the formulation.

RELATED APPLICATIONS

The present application is a continuation of U.S. patent applicationSer. No. 10/494,758 filed on Nov. 30, 2004 (Attorney Docket No.FMG001-001), which is the

U.S. National Phase of international PCT Application No.PCT/BR2002/000156 filed on Nov. 5, 2002,which claims priority toBrazilian Application No. PI 0105509-7 filed on Nov. 5, 2001, thecontents of which are hereby incorporated by reference.

FIELD OF THE INVENTION

The present invention is characterized by the process of preparation offormulations of the heptapeptide Angiotensin-(1-7) and its similar ones,agonists and antagonists, using preferentially the cyclodextrins, andderivatives, liposomes, and biodegradable polymers, and/or mixtures ofthose systems and/or of the derived products. It is also characterizedby the identification of the ligand-receptor interaction between theG-coupled receptor, MAS, and angiotensin-(1-7) and its analogues usingor not encapsulated formulations, as a target for research andtherapeutic interventions in cardiovascular, renal, reproductive,dermatological, tumoral, neurological and blood diseases.

BACKGROUND OF THE INVENTION

In most countries of the world 15% to 25% of the adult populationpresents high arterial blood pressure (MacMahon S. et. al. Bloodpressure, stroke, and coronary heart disease. Lancet 335:765-774, 1990).The cardiovascular risk increases with the level of arterial pressure.The more high the arterial pressure bigger are the risk of cerebralvascular accidents and coronary events. Hypertension, considered themain responsible cause for coronary, cerebral and renal vasculardiseases, is the main cause of death and incapability among adults.

The heart failure is worldwide the main cause of hospitalization ofpatients in the age group of 60 to 80 years old. The aging of thepopulation is already an important factor for the increase in theincidence of heart failure: while 1% of the individuals in the age rangeof 25 to 54 years old present heart failure, among the more seniors theincidence is very larger, reaching about 10% in those individuals withmore than 75 years (Kaannel W. B. et. al. Changing epidemiologicalfeatures of cardiac failure. Br. Heart J 1994; 72 (suppl):S3-S9).

The heart failure by its clinical characteristic is a restrictivedisease, that reduces the quality of the patients' life with itsworsening and, in the advanced forms, is characterized as a maligndisease with mortality rate above 60% in the first year, even nowadays(Oliveira, M. T. Clinical characteristics and patients' prognostic withadvanced congestive heart failure. Faculty of Medicine, USP 1999). It isestimated that these days there are more than 15 million of individualsaffected only in the industrialized world and that only in the USA, forexample, the number of cases has increased 450% among 1973-1990 (Kannel,W. B. et al. Changing epidemiological features of cardiac failure, Br.Hear J 1994; 72 (suppl 3): S3-S9).

Hypertension is a complex, multifactorial, of high prevalence diseaseresponsible for countless deleterious effects and high morbidity(Kaplan, N. M. Blood pressure the cardiovascular risk factor: preventionant treatment. JAMA. 275:1571-1576, 1996). Several studies aimed for theevaluation of the effectiveness of its control in the population ingeneral and in special groups have been developed, in order to betterunderstand its course. The control of the normal level of blood pressurewithout wide intervention with no pharmaceutical drugs of the associatedrisk factors (such as, diabetes, obesity, smoke) can reduce or evenavoid the benefits of the long term treatment of arterial hypertensionin the reduction of the mortality, in general, as for coronary disease(Wilson, P. W. et. al. Hypertension, the risk factors and the risk ofcardiovascular disease. Raven Press. 94-114).

Hypertension is the pathology that more contributes to thecardiovascular atherosclerosis (The fifth Report of the Joint NationalCommittee on detection, evaluation, and treatment of High BloodPressure. National Institute of Health (VJNC). Arch. Intern. Med.153:154-181, 1994). According to statistics, of each four Americans, oneis, or will be, hypertensive, and it is estimated 4,78 million peoplewith heart failure. Every year 400 thousand new cases are diagnosed,provoking 800 thousand hospitalizations, with an expense of US$ 17,8billion of dollars in the treatment.

In Brazil, data from the National System of Health (SUS) show that, in1997, heart failure was the main cause of hospitalizations among theheart diseases, costing about R$ 150 million for the government in thetreatment, corresponding to 4,6% of the whole expense with health(Filho, Albanesi F. Heart failure in Brazil. Arq. Bras. Cardiol,71:561-562, 1998).

The renin-angiotensin system (RAS) is responsible for the regulation ofthe arterial pressure, cardiovascular homeostase and hydroelectrolitebalance, as in physiological as in pathological conditions (Krieger, E.M.; Santos, R. A. S. Angiotensins—physiologic aspects. Hypertension,1:7-10, 1998). Angiotensin II (Ang II) is the main peptide of RAS,possessing several actions: vasopressor, stimulator of the synthesis ofadrenal steroids, proliferative effect (fibroblasts, smooth muscle ofthe vasculature) and hypertrophic (cardiomyocytes). Its formationpathways involve the production of angiotensinogen from the liver andrenin production in the juxtaglomerular apparatus. Those substances arereleased in the blood where, the angiotensinogen is hydrolysed by renin,forming Ang I, that, is converted to Ang II, mainly the lungcirculation, by the angiotensin converting enzyme (ACE) and it willoriginate Ang II. This last peptide will act in target-organs distantfrom the place of its production (Krieger, E. M.; Santos, R. A. S.Angiotensins—physiologic aspects. Hypertension, 1: 7-10, 1998).

Recently it was discovered that in parallel to the circulating RAS, thatgenerate Ang II in the circulation, there are independent systems thatgenerates Ang II in different tissues probably for a local action. Allthe components of the RAS are found in the walls of the blood vessels,in the uterus, in the exocrine portion of the pancreas, eyes, heart,adrenal cortex, testicle, ovaries, anterior and median lobes of thehypophise, pineal and brain. The function of those tissue RAS are notvery well understood. (Ardaillou, R.; Michel, J. B. The relative rolesof circulating and tissue renin-angiotensin systems. Nephrol. Dial.Transplant., 14:283-286, 1999). The local actions of RAS can occur atthe level of the cell that produces the peptide (as an autocrine orintracrine function), or on adjacent cells (as a paracrine function), orin distant sites from the production area (endocrine function).

Recent observations indicate that important peripheral and cerebralactions of the RAS can be mediated by smaller sequences of theangiotensinergic peptides, including Angiotensin-III [Ang-(2-8)],Angiotensin-IV [Ang-(3-8)] and Angiotensin-(1-7). We can consider thatboth Angiotensin-I (Ang-(1-10)] and Angiotensin-II [Ang-1-8)] can suffera biotransformation process, generating a whole “family” of biologicalactive angiotensin peptides. (Santos, R. A. S.; Campagnole-Santos, M.J.; Andrade, S. P. Angiotensin-(1-7): an update. Regulatory Peptides,91:45-62, 2000).

Angiotensin-(1-7) is a biologically active peptides of the angiotensin“family”, being formed by a pathway indenpendent of the ACE. Ang Iprocessing by endopeptidases or Ang II by prolil-peptidases orcarboxy-peptidases can generate the heptapeptide, Ang-(1-7). Ang-(1-7)can be hydrolysed by amino-peptidases generating Ang-(2-7) andAng-(3-7). The hydrolysis of Ang-(1-7) by ACE originates Ang-(1-5)(Santos, R. A. S.; Campagnole-Santos, M. J.; Andrade, S. P.Angiotensin-(1-7): an update. Regulatory Peptides, 91:45-62, 2000).

Ang-(1-7) and Ang II are the main efectors of the RAS. However, twoimportant characteristics differentiate Ang-(1-7) from Ang II: the firstone possesses highly specific biological actions and its formationpathway is independent of ACE (Santos, R. A. S.; Campagnole-Santos, M.J.; Andrade, S. P. Angiotensin-(1-7): an update. Regulatory Peptides,91:45-62, 2000).

The primary objective of hypertension treatment not only seeks for thefall expenses, but as well as the prevention of end-organ damages,through modifications of life quality and the use of medications, whennecessary (The Fifth Report of The Joint National Committee ondetection, evaluation, and treatment of High Blood Pressure. NationalInstitute of Health (VJNC). Arch. Intern. Med. 153:154-181, 1994).

The use of anti-hypertensive drugs is indicated when patients do notrespond to the alterations in lifestyle for a period of three to sixmonths, and in the presence of end-organs damage (left ventricularhypertrophy, myocardic ischemia, stroke, or hypertensive retinopathy).All patients with systolic arterial pressure above 180 mmHg or diastolicarterial pressure above 110 mmHg should be submitted to pharmacologicaltreatment, independent of the presence of another (Report the CanadianHypertension Society. Consensus Conference, 3. Pharmacological treatmentof essential hypertension. Xan. Med. Assoc. J. 149 (3): 575-584, 1993).

During the years 70 and 80, however, the anti-hypertensives became animportant tool for the treatment of the high arterial pressure (Mnard,J. Anthology of renin-angiotensin system: The one hundred referenceapproach to angiotensin antagonist II. J. Hypertension 11 (suppl 3):S3-S11, 1993). During the last four decades, the pharmacologicalresearch produced new classes of drugs to treat the hypertension: thediuretics in the sixties, the beta-blockers in the seventies, thecalcium channel blockers, the angiotensin II antagonists and the ACE ininhibitors.

The diuretics can be divided in three categories: thazidics, loopdiuretics and the potassium savers. The thazidics and analogues includechlorothiazides and hydroclorotiazide, which induce in the first days oftreatment a 10-15% of decrease in the arterial pressure mainly due to adecrease in the extracellular volume and an increase in the diuresis andnatriuresis. After six months, the blood volume and cardiac outputreturn to baseline levels and the decrease in arterial pressure ismaintained by a decrease in peripheral vascular resistance (Frolich, E.Current Approaches in the treatment of Hypertension, 405-469). Thesedrugs are habitually used as monotherapy and the give best results inblack patients and, at low doses, in the old patients. They have ascollateral effects: increase in peripheral resistance to insulin,increase in triglicerides levels, increase in LDL, hypocalcemia,hyperucemia. Among the loop diuretics are furosemide, bumetamide andtrianterene, and they are much more potent than the thiazides. They actpredominantly in the medullar and cortical portions of the Henle loop.They present the same collateral effects of the thiazides. The potassiumsavers, however, which include amolonide, trianterene andsperonolactones are drugs with weak diuretic action and rarely usedalone.

The betablockers, such as Atenolol and Nadolol, are classified in beta-1and beta-2 and the mechanism of action are not completely defined. Theypresent as collateral effects: alteration in the response to insulin,prolongation of the hypoglycemic coma, increase in triglicerides levelsand increase in creatinine due to a decrease in renal flow.

The calcium channel blockers are being used for at least 25 years(Frolich, E. D. Current Approaches in the Treatment of Hypertension,405-469, 1994). They can be classified in two major groups, according toits pharmacological actions: those that have larger action in theconduction of the stimulus, such as Verapamil and Diltiazem, and thosethat present a predominant vasodilator action, as those derived fromdiidropirinicos (Nifedipine and others) (Frolich, E. D., Hypertension.Adult Clinical Cardiology Self Assessment Program (ACCSAP), 6: 3-19,1995). They present as collateral effects edema of inferior members andtachycardia.

The main action of the ACE inhibitors is to inhibit the conversion ofangiotensin I in to angiotensin II. Thus, the essentiallyvasoconstrictor actions of angiotensin II are minimized. Teprotide, thefirst ACE inhibitor clinically used, exerted its anti-hypertensiveaction only when it was administered by intravenous route, because itwas inactive when given orally, what have limited its employment. It isknown now that ACE is an enzyme with multiple actions, i.e., that itacts in several substrate. Besides acting as a dipeptidase in theangiotensin I and in the bradykinin, it is also capable of hydrolysingpeptidic chains of the natriuretic peptide, indicating that the enzymecan act in several tissues. ACE has an important role in theinactivation of circulating and tissue Ang-(1-7). The circulatingconcentration of this peptide is similar to Ang II concentration and ithas been shown that it increases after inhibition of ACE. This increasecan be due to both the increase in its precursor (Ang I) and thedecrease in its degradation by ACE (Santos, R. A. S.; Campagnole-Santos,M. J.; Andrade, S. P. Angiotensin-(1-7): an update. Regulatory Peptides,91:45-62, 2000). The ACE inhibitors are excellent when administered asmonotherapy, since they induce a relatively fast fall in arterialpressure in 60 to 70% hypertensive patients. (Ganong, W. Neuropeptidesin cardiovascular control. J. Hypertens 2 (suppl 3): 15-22, 1984). Inaddition, they are in general well tolerated, but its use can causecollateral effects and adverse reactions, some of which are relativelyserious, among them angiodema, cutaneous eruptions and dry coughs (8 to10%).

The first attempts to develop Ang II antagonists are from the beginningof the 70′s decade and they were concentrated on the development ofpeptides similar to Ang II. The first antagonist was saralasina,1-sarcosina, 8-isoleucina angiotensin II, that was followed by others.However, they did not have clinical acceptance, because they presentedpartial agonist activity. In 1982 were developed the first two selectiveantagonists for the AT₁ receptor of non-peptide characteristic (S-8307and S8308). However, eventhough they were highly specific and withoutagonist activity, they presented a weak bind to Ang II'S receptors. Witha series of modifications in the molecular structure of those twoprecursors, to improve the potency and to retain the selectivity and toreach the pharmacokinetis properties, a new product of orally active,potent and of high specificity was developed, Losartan. Starting fromthen, many other antagonists of non-peptidic origin were developed, suchas Candesartan, Irbesartan, Valsartan, Telmisartan, Eprosartan,Tasosartan and Zolasartan.

Angiotensin-(1-7), (Asp-Arg-Val-Tyr-Ile-His-Pro) and its derivative one[Sar¹]-Ang-(1-7) antagonize Ang II pressor effect in man (Ueda S,Masumori-Maemoto S, Ashino K, Nagahara T, Gotoh and, Umemura S, Ishii M.Angiotensin-(1-7) attenuates vasoconstriction evoked by angiotensin IIbut not by noradrenaline in man. Hypertension 2000; 35:998-1001) andmice (Bovy P R, Trapani A J, McMahon E G, Palomo M. The carboxy-terminustruncated analogue of angiotensin II [Sar¹]-angiotensin II-(1-7)-amide,provides an entry to the new class of angiotensin II antagonists. J MedChem. 1989; 32:520-522). The contraction produced by Ang II in isolatedarteries of rabbits and humans is also reduced by angiotensin-(1-7)(Bovy P R, Trapani A J, McMahon E G, Palomo M. The carboxy-terminustruncated analogue of angiotensin II [Sar¹] angiotensin II-(1-7)-amide,provides an entry to the new class of angiotensin II antagonists. J MedChem. 1989; 32:520-522. Roks A J, Van-Geel P P, Pinto Y M, Buikema H,Henning R H, of Zeeuw D, van-Gilst W H. Angiotensin-(1-7) is a modulatorof the human renin-angiotensin system. Hypertension 1999;34(2):296-301).

Until very recently, the receptor(s) responsible for the transduction ofthe Ang-(1-7) response had not been identified and many possibilitieswere raised regarding Ang-(17) signal transduction. The first evidencefor the existence of different receptors and/or different mechanisms ofsignal transduction for Ang-(1-7) effects was based on the demonstrationthat several Ang-(1-7) actions are different and even opposite fromthose ascribed for Ang II. Recently, the heptapeptide D-[Ala⁷]-Ang-(1-7)(A-779) was characterized as a potent antagonist for Ang-(1-7) effects(Santos R A S, Campagnole-Santos M J, Baracho N C V, Fontes M A P, SilvaL C S, Neves L A A, Oliveira D R, Caligiorne S M, Rodrigues A R V,Gropen Jr. C, Carvalho W S, Silva A C S, Khosla M C. Characterization ofthe new angiotensin antagonist selective goes angiotensin-(1-7):Evidence that the actions of angiotensin-(1-7) it plows mediated byspecific angiotensin receptors. Brain Res. Bull. 1994;35:293-299). Theresults of that study indicated that this analogue is a selectiveantagonist of Ang-(1-7) without demonstrating agonist activity inseveral biological preparations. A-779 was shown to potently antagonizethe antidiuretic effect of Ang-(1-7) in rats with water overload. Thevasodilatation produced by Ang-(1-7) in the afferent arterioles ofrabbits, the Ang-(1-7) pressor effect in the RVLM, the vasodilationproduced in the mesenteric microcirculation in vivo are completelyblocked by the administration of A-779, and are not affected by theselective Ang II antagonists. Other studies using bovine endothelialcells, dog coronary arteries, SHR aorta, human epithelial fibroblasts,human heart fibroblasts and kidney slices have supported the evidencesfor the existence of specific receptors of Ang-(1-7) that can be blockedby the A-779. (Santos, R A S; Campagnole-Santos, M J.; Andrade, S P.Angiotensin-(1-7): an update. Regulatory Peptides, 91:45-62, 2000).

A-779 and its analogues such as Sarl-D-Ala 7-Ang-(1-7) (Bovy P R,Trapani A J, McMahon E G, Palomo M. THE carboxy-terminus truncatedanalogue of angiotensin II [Sar1] angiotensin II-(1-7)-amide, providesan entry to the new class of angiotensin II antagonists. J Med Chem.1989; 32:520-522.), and the D-Pro7-Ang-(1-7) (Naves-Santos, V., Khosla,M. C., Oliveira, R. C., Campagnole-Santos, M. J., Lima, D. X., Santos, RA S. Selective inhibition of the effect central pressor ofangiotensin-(1-7) for its similar one [D-Pro7]-angiotensin-(1-7). XIReunio Annual of the Federation of Society of Experimental Biology,1996, Caxambu, M G) and others can serve extremely as tools to elucidatebiological effects of Ang-(1-7).

It has been demonstrated that Ang-(1-7) acts inside the RAS as acontraregulatory peptide of this system, acting at multiple points(Ferrario C M, Chappell M C, Dean R H, Iyer S N. Novel angiotensinpeptides regulate blood pressure, endothelial function, and natriuresis.J Am Soc Nephrol. 1998; 9: 1716-1722. Santos, R. Campagnole-Santos, M J,Andrade, S P. Angiotensin-(1-7): an update. Regulatory Peptides,91:45-62, 2000. Heringer-Walther S, Batista E N, Walther T, Khosla M C,Santos R A S, Campagnole-Santos M J. Baroreflex improvement in SHR afterACE inhibitors involves angiotensin-(1-7). Hypertension, 37: 1309-1313,2001).

Ang-(1-7) decreases angiogenesis and cellular proliferation (Machado, RD P, Santos, R A S, Andrade, S P. Mechanisms of angiotensin-(1-7)induced inhibition of angiogenesis. Am J Physiol, 280: 994-1000, 2001.Rodgers K, Xiong S, Flix J, Rotates N, Espinoza T, Maldonado S, DizeregaG. Development of angiotensin-(1-7) the in the agent to acelerate dermalrepair. Wound Repair Regen, 9: 238-247, 2001) presenting therefore apotential for the treatment of lesions. Ang-(1-7) can act as an ACEinhibitor in the amino-terminal domain of the enzyme, in which it actsas substrate, as well in the c-terminal domain in which it acts as aninhibitor (Deddish P A, Marcic B, Jackman H L, Wang H Z, Skidgel R A,Erdos E G. N-domain-specific substrate and C-domain inhibitors ofangiotensin-converting enzyme: angiotensin-(1-7) and keto-ACE.Hypertension. 1998; 31:912-917. Tom B, Of Vries R, Saxena P R, Danser AH J. Bradykinin potentiation by angiotensin-(1-7) and ACE inhibitorscorrelates with ACE C- and N-domain blockade. Hypertension, 38: 95-99,2001). Its IC50 for inhibition of ACE is approximately 1 micromolar(Chappell M C, Pirro N T, Sykes T H E, Ferrario C M. Metabolism ofangiotensin-(1-7) by angiotensin-converting enzyme. Hypertension. 1998;31(part 2):362-367. Paula, R D, Lima, C V, Britto, R R,Campagnole-Santos, M J, Khosla, M C, Santos, R A S. Potentiation of thehypotensive effect of bradykinin by angiotensin-(1-7)-related peptides.Peptides, 20:493-500, 1999. Deddish P A, Marcie B, Jackman H L, Wang HZ, Skidgel R A, Erdos E G. N-domain-specific substrate and C-domaininhibitors of angiotensin-converting enzyme: angiotensin-(1-7) andketo-ACE. Hypertension, 31:912-917, 1998).

In addition to the ACE inhibitory activity, Ang-(1-7) inhibits the AngII actions by two different mechanisms: 1) competing for the ligation onAT₁ receptors (Bovy P R, Trapani A J, McMahon E G, Palomo M. Thecarboxy-terminus truncated analogue of angiotensin II [Sar¹]-agiotensinII-(1-7)-amide, provides an entry to the new class of angiotensin IIantagonists. J Med Chem. 1989; 32:520-522. Ueda S, Masumori-Maemoto S,Ashino K, Nagahara T, Gotoh A N D, Umemura S, Ishii M. Angiotensin-(1-7)attenuates vasoconstriction evoked by angiotensin II but not bynoradrenaline in man. Hypertension 2000; 35:998-1001. Roks A J, Van-GeelP P, Pinto Y M, Buikema H, Henning R H, deZeeuw D, van-Gilst W H.Angiotensin-(1-7) is a modulator of the human renin-angiotensin system.Hypertension 1999; 34(2):296-301. Rowe B P, Saylor D L, Speth R C,Absher D R. Angiotensin-(1-7) binding at angiotensin II receptors in therat brain. Regul Pep. 1995; 56(2):139-146. Mahon J M, Carrr R D, Nicol AK, Hendersn I W. Angiotensin-(1-7) is an antagonist at the type 1angiotensin II receptor. J Hypertension 1994; 12:1377-1381), and 2)altering the signalling mechanisms of Ang II effects, possibly byaltering the availability of intracellular calcium (Chansel D,Vandermeerch S, Andrzej T, Curat C, Ardaillou R. Effects of angiotensinIV and angiotensin-(1-7) on basal angiotensin II-stimulated cytosolicCa+2 in mesangial cells. Eur J Pharmacol. 2001; 414:165-175). A thirdmechanism for which Ang-(1-7) can antagonize the Ang II deleteriouseffects on the cardiovascular system are the potentiation of thebradykinin effects (Paula, R D; It Rasps, C V, Khosla, M C, Santos, R AS. Angiotensin-(1-7) potentiates the hypotensive effect of bradykinin inconcious rats. Hypertension, 26: 1154-1159, 1995. Li P, Chappell M C,Ferrario C M, Brosnihan K B. Angiotensin-(1-7) augmentsbradykinin-induced vasodilation by competing with ACE and releasingnitric oxide. Hypertension. 1997; 29 (part 2):394-400).

Bradykinin is an endogenous peptide with potent vasodilatatory action(Rocha and Silva, M, Beraldo, W T, Rosenfeld, G. Bradykinin, thehypotensive and smooth muscle stimulating factor releases from shapesglobulin by snake venoms and by trypsin. Am. J. Physiol. 156, 261-273,1949). It has also been described beneficial actions of bradykinin inthe heart (Linz W, Wohlfart P, Scholkens B A, Malinski T, Wiemer G.Interactions among ACE, kinins and NO. Cardiovasc Res. 1999;43:549-561). Ang-(1-7) potentiate the effects of bradykinin, in vessels(Paula, R. D.; Lima, C. V.; Khosla, M. C.; Santos, R. A. S.Angiotensin-(1-7) potentiates the hypotensive ffect of bradykinin inconcious rats. Hypertension, 26: 1154-1159, 1995. Li P, Chappell M C,Ferrario C M, Brosnihan K B. Angiotensin-(1-7) augmentsbradykinin-induced vasodilation by competing with ACE and releasingnitric oxide. Hypertension. 1997; 29 (part 2):394-400), in the heart(Almeida, A P, Frbregas, B C, Madureira, M M, Santos, R J S,Campagnole-Santos, M J, Santos, R A S. Angiotensin-(1-7) potentiates thecoronary vasodilatory effect of bradykinin in the isolated rat heart.Braz. J. of Medical and Biological Research, 33: 709-713, 2000).

A particular drug could be chemically modified in order to alter itsproperties such as biodistribution, pharmacokinetics and solubility.Various methods have been used to increase the solubility and stabilityof drugs, among them the use of organic solvents, their incorporationwithin emulsions or liposomes, the adjustment of pH, their chemicalmodifications and their complexation with the cyclodextrins.

The cyclodextrins are oligosaccharides cyclic family, which include six,seven or eight units of glucopyranose. Due to sterics interactions, thecyclodextrins, CD's, form a cycle structure in the shape of a (conetruncado) with an internal cavity apolar. Those are compounds chemicallystable that can be modified in a regioselective way. The cyclodextrinshosts form complexes with various hydrophobic guests in their cavity.The CD's have been used for the solubilization and encapsulation of thedrugs, perfumes and fragrances as described by Szejtli, J., ChemicalReviews, (1998), 98, 1743-1753. Szejtli, J., J. Mater. Chem., (1997), 7,575-587.

According to detailed studies of toxicity, mutagenecity, teratogenecityand carcinogenecity about the cyclodextrins, described in [Rajewski, R.A., Stella, V., J. Pharmaceutical Sciences, (1996), 85, 1142-1169],these are presented with low toxicity specially of the(hydroxypropyl-β-cyclodextrin, as reported in Szejtli, J. Cyclodextrins:properties and applications. Drug investing., 2(suppl. 4):11-21, 1990.Except for some high concentrations of some derivates which cause harmto the erythrocytes, these products in general are not harmful to thehealth. The use of cyclodextrins as additives in foods has already beenauthorized in countries such as Japan and Hungary, and for more specificapplications, in France and Denmark. Besides this, they are obtainedfrom a renewable source of degradation of the amide. All thesecharacteristics are a high motivation for the research findings of newapplications. The structure of the molecule of CD is similar to a conetruncate one, of Cn approximately symmetry. The primary hydroxilas arelocated in the narrowest side of the cone by the intramolecular hydrogenbonds, this element is flexible enough to allow a considerable deviancein the regular shape.

The known cyclodextrin derivatives can be classified according to theirpolarity, size, biological activity, etc. As for their practical usesare classified as follows: 1. Carriers (solubilizers, stabilizers) forbiologically active substances; 2. Enzyme models; 3. Separating agents(for chromatography or batch-processes); 4. Catalysts and additives (asdetergents, viscosity modifiers, etc), L. Szente and J. Szejtli, Adv.Drug Deliv. Rev. 36 (1999), 17. The CD's are moderately soluble inwater, methanol and ethanol and readily soluble in polar solvents, suchas the dimethyl sulfoxide, dimethyl formamide, N,N-dimethyl acetamideepiridine.

Numerous research works exist in the literature about the effects of theincrease of solubility in water of the guests little soluble in water,using the cyclodextrins through the using compounds of inclusion weredescribe in Szejtli, J., Chemical Reviews, (1998), 98, 1743-1753.Szejtli, J., J. Mater. Chem., (1997), 7, 575-587.

In order to design a drug delivery system (DDS) various kinds of highperformance carrier materials are being developed to deliver thenecessary amount of drug to the targeted site for a necessary period oftime, both efficiently and precisely. Cyclodextrins, biodegradable ornon biodegradable polymers, liposomes, emulsions, multiple emulsions arepotential candidates for such a role, because of their ability to alterphysical, chemical, and biological properties of guest molecules

Besides the cyclodextrins, a number of drug delivery systems have beeninvestigated, including polymer microcapsules, microparticles, liposomesand emulsion. Many of these are prepared from synthetic biodegradablepolymers such as polyanhydrides and poly(hydroxy acids). In thesesystems the drugs incorporate in a polymeric microspheres, which releasethe drug inside the organism, in small and controlled daily doses,during days, months or until years.

Several polymers already were tested in controlled release systems. Suchas: polyuretans for its elasticity, polysiloxans or silicons for being agood one insulating, polymethyl-methacrylate for its physical force,polyvinyl alcohol for its hydrophobicity and resistance, polyethylenefor its hardness and impermeability (Gilding, D. K. Biodegradablepolymers. Biocompat. Clin. Impl. Mater. 2:209-232, 1981). Biodegradablepolymers and biocompatible polymers, have been extensively investigatedas vehicle for controlled release systems due to their ability toundergo surface degradation. These kind of polymers can be chose from:poly(hydroxy-ethylmethacrylate), polyacrylamide, polymer from lacticacid (PLA), from glicolic acid (PGA), and the respective onesco-polymers, (PLGA) and the poly(anhydrides), as described by Tamada andLanger, J. Biomater. Sci. Polym. Edn, 3(4):315-353.

A formulation of the present invention can also include other componentssuch as a pharmaceutical acceptable excipient. For example, formulationof the present invention can be formulated in an excipient that theanimal to be protected can tolerate. Excipients can also contain minoramounts of additives, such as substances that enhance isotonicity andchemical stability of buffers. Standard formulation can either be liquidinjectables or solids which can be taken up in a suitable liquid as asuspension or solution for injection or oral formulation. Suitablecontrolled release vehicles include, but are not limited to,biocompatible polymers, other polymeric matrices, capsules,microcapsules, nanocapsules, microparticles, nanoparticles, boluspreparations, osmotic pumps, diffusion devices, liposomes, lipospheresand transdermal delivery systems, implantable or not.

In the last years, several systems of drugs delivery systems have beenstudied to improve the drug absorption, to increase the drug stabilityand target it to a certain cell population. These studies led to thedevelopment of several products based on cyclodextrins, emulsions,liposomes and polymers for drug carrying and delivering. Theseformulations can be administered through intramuscular, intravenous,subcutaneous injection, oral application, inhalation or devices that canbe implanted.

Liposomes are lipid vesicles that include aqueous internal compartmentsin which molecules, for example drugs, are encapsulated with theobjective of reaching a controlled release of the drug afteradministration in individuals.

Many different techniques have been proposed for the preparation ofliposomes [U.S. Pat. No. 4,552,803, Lenk; U.S. Pat. No. 4,310,506,Baldeschwieler; U.S. Pat. No. 4,235,871, Papahadjopoulos; U.S. Pat. No.4,224,179, Schneider; U.S. Pat. No. 4,078,052, Papahadjopoulos; U.S.Pat. No. 4,394,372, Tailor; U.S. Pat. No. 4,308,166, Marchetti; U.S.Pat. No. 4,485,054, Mezei; and U.S. Pat. No. 4,508,703, Redziniak;Woodle and Papahadjopoulos, Methods Enzymol. 171:193-215 (1989)].Unilamellar vesicles display a single membrane [Huang, Biochemistry8:334-352 (1969)] while multilamellar vesicles (MLVs) have numerousconcentric membranes [Bangham et al., J. Mol. Biol. 13:238-252 (1965)].The procedure of Bangham [J. Mol. Biol. 13:238-252 (1965)] produces“ordinary MLVs”, that present unequal solute distributions among theaqueous compartments and, consequently, differences of osmotic pressure.Lenk et al. (U.S. Pat. No. 4,522,803; U.S. Pat. No. 5,030,453 and U.S.Pat. No. 5,169,637), Fountain et al. (U.S. Pat. No. 4,588,578), Culliset al. (U.S. Pat. No. 4,975,282) and Gregoriadis et al. (Pat. W.O.99/65465) introduced methods for the preparation of MLVs that presentsubstantially equal solute distributions among the compartments. Similarsolute distributions among the different compartments mean a larger drugencapsulation efficiency as well as smaller differences of osmoticpressure that turns these MLVs more stable than ordinary MLVs.Unilamellar vesicles can be produced by sonication of MLVs[Papahadjopoulos et al. (1968)] or by extrusion through polycarbonatemembranes [Cullis et al. (U.S. Pat. No. 5,008,050) and Loughrey et al.(U.S. Pat. No. 5,059,421)].

Satisfactory lipids include for example, phosphatidylcholine,phosphatidylserine, phosphatidylglycerol, cardiolipin, cholesterol,phosphatidic acid, sphingolipids, glycolipids, fatty acids, sterols,phosphatidylethanolamine, polymerizable lipids in their polymerized ornon-polymerized form, mixture of these lipids.

The composition of the liposomes can be manipulated such as to turn themspecific for an organ or a cell type. The targeting of liposomes hasbeen classified either on the basis of anatomical factors or on thebasis of the mechanism of their interaction with the environment. Theanatomical classification is based on their level of selectivity, forexample, organ-specific or cell-specific. From the point of view of themechanisms, the targeting can be considered as passive or active.

The passive targeting exploits the natural tendency of conventionalliposomes to be captured by the cells of the reticulo-endothelialsystem, i.e. mainly the fixed macrophages in the liver, spleen and bonemarrow. Sterically stabilized liposomes (also well-known as“PEG-liposomes”) are characterized by a reduced rate of elimination fromthe blood circulation [Lasic and Martin, Stealth Liposomes, CRC Press,Inc., Boca Raton, Fla. (1995)]. PEG-liposomes present a polyethyleneglycol polymer conjugated to the head group of some phospholipid thatreduces their interaction with plasma proteins, such as opsonins, andreduces the rate of their uptake by cells. The resulting steric barrierallows these liposomes to remain for a longer period of time within thecirculation than conventional liposomes [Lasic and Martin, StealthLiposomes, CRC Press, Inc., Boca Raton, Fla. (1995); Woodle et al.,Biochim. Biophys. Acta 1105:193-200 (1992); Litzinger et al., Biochim.Biophys. Acta 1190:99-107 (1994); Bedu Addo, et al., Pharm. Res.13:718-724 (1996)]. The drug encapsulation within PEG-liposomes hasresulted in the improvement of the effectiveness of manychemotherapeutic agents [Lasic and Martin, Stealth liposomes, CRC Press,Inc., Boca Raton, Fla. (1995)] and bioactive peptides [Allen T. M. In:Liposomes, New Systems, New Trends in their Applications (F. Puisieux,P. Couvreur, J. Delattre, J. -P. Devissaguet Ed.), Editions de la Sant,France, 1995, pp. 125].

Studies in this area demonstrated that different factors affect theeffectiveness of PEG-liposomes. Ideally, the diameter of the vesiclesshould be below 200 nm, the number of units in PEG of approximately2,000 and the proportion of Pegylated lipid from 3 to 5 mol % [Lasic andMartin, Stealth Liposomes, CRC Press, Inc., Boca Raton, Fla. (1995);Woodle et al., Biochim. Biophys. Acta 1105:193-200 (1992); Litzinger etal., Biochim. Biophys. Acta 1190:99-107 (1994); Bedu Addo et al., Pharm.Res. 13:718-724 (1996)].

The active targeting involves alteration of liposomes through theirassociation with a ligand, such as a monoclonal antibody, a sugar, aglycolipid, protein, a polymer or by changing the lipid composition orthe liposome size to target them to organs and cells different fromthose which accumulate conventional liposomes.

Liposome-based vehicles have been proposed for a large variety ofpharmacologically active substances, including antibiotics, hormones andantitumoral agents [Medical applications of liposomes (D. D. Lasic, D.Papahadjopoulos Ed.), Elsevier Science B. V., Holland, 1998].

Ang-(1-7) and its analogues have great potential for study and treatmentof several diseases including cardiovascular disorders. Anotherimportant aspect related RAS is related to the clear need ofamplification of the knowledge about its physiologic actions that canpropitiate the development of new therapeutic strategies. However, theconventional way of administration of most of the drugsanti-hypertensive especially biologically active peptides, as theangiotensins and derivatives, suffers limitations due to the shorthalf-life of peptides.

In that sense, the present invention is characterized by the use ofliposomes, cyclodextrins and biodegradable polymers as controlledrelease systems of the angiotensins and derivatives to increase theirbioavailability, the duration and intensity of their biological effects.

The formulation of the present invention is characterized by the use ofthe mixture of excipients pharmaceutically acceptable for Ang-(1-7)and/or analogues. Excipients examples include water, saline solution,buffered phosphate solutions, the solution of Ringer, dextrose solution,the solution of Hank, biocompatible saline solutions with or withoutpolyethylene glycol. Non aqueous vehicles, as fixed oils, sesame oil,ethyl-oleate, or triglicerides can also be used. Other usefulformulations include agents capable to increase the viscosity, ascarboxymetilcelulose of sodium, sorbitol, or dextran

The excipients can also contain smaller amounts of additives, such assubstances that increase isotonicity and chemical stability of substanceor buffers. Examples of buffers include phosphate buffer, bicarbonatebuffer and Tris buffer, while examples of preservatives includetimerosal, m- or o-cresol, formalin and benzyl-alcohol. The formulationstate can be liquid or solid. In the case of a non-liquid formulation,the excipients can include dextrose, human serum albumin, preservatives,etc. for which water or sterile saline solution can be added before theadministration.

The present invention is also characterized by the preparation ofcontrolled release systems containings Ang-(1-7) and/or its analoguesfor interaction ligand-receptor with the G Protein-coupled receptor,MAS. Satisfactory systems of controlled release include, but are notlimited to, the cyclodextrins, biocompatible polymers, biodegradablepolymers, other polymeric matrixes, capsules, micro-capsules,microparticles, bolus preparations, osmotic pumps, diffusion devices,liposomes, lipospheres, and systems of transdermic administration. Othercompositions of controlled release of the present invention includeliquids that, when submitted the temperature changes, form a solid or agel in situ.

The MAS receptor (Young, D., Waitches, G., Birchmeier, C., Fasano, O.,and Wigler, M. (1986). Isolation and characterization of a new cellularoncogene encoding a protein with multiple potential transmembranedomains. Cell 45: 711-719) was initially described as an angiotensin IIreceptor (Jackson, T. R., Blair, A. C., Marshall, J., Goedert, M. &Hanley, M. R. The MAS oncogene encodes an angiotensin receptor. Nature335, 437-440 (1988)), however subsequent studies showed that thishypothesis was not right (Ambroz, C., Clark, A. J. L. & Catt, K. J. TheMAS oncogene enhances angiotensin-induced [Ca2+]i responses in cellswith pre-existing angiotensin II receptors. Biochem. Biophys. Acta 1133,107-111 (1991)). This protein is expressed in the brain (Bunnemann, B.,Fuxe, K., Metzger, R., Mullins, J., Jackson, T. R., Hanley, M. R. &Ganten, D. Autoradiographic localization of MAS proto-oncogene mRNA inadult rat brain using in situ hybridization. Neurosci. Left. 114,147-153 (1990)) and in other tissues. There is no description in theliterature of an interaction of MAS with angiotensin-(1-7) or itsanalogues.

The present invention is characterized by the obtention of systems ofcontrolled release of the heptapeptide Angiotensin-(1-7) and/or itsderivatives, using the cyclodextrins and/or its derivatives, thatdecrease the degradation of the peptide in the treatmentgastrointestinal (TGI), meaning larger biodisponibility of the peptidein the biological system. The present invention it is characterized bythe obtention of controlled release systems of the heptapeptideAngiotensin-(1-7) and/or of its analogues, using biodegradable polymers,liposomes or mixtures of those systems with cyclodextrins, whichincrease the biodisponibility of the peptides.

Until the present, any application using the heptapeptideAngiotensin-(1-7) or its analogues, agonists and antagonists associatedto the cyclodextrins or theirs derivatives, to biodegradable polymers orto liposomes, was not described.

The present invention can be understood better through the followingexamples:

EXAMPLE 1

This example describes the preparation of Ang-(1-7) in the encapsulatedform in sterically stabilized liposomes and the improvement of thebioavailability of Ang-(1-7) when administered in that form.

The encapsulation of Ang-(1-7) in liposomes was performed according to

Kirby and Gregoriadis [Biotechnology 2:979-984 (1984)] and was followedby the extrusion of the liposome suspension through polycarbonatemembranes with a pore size of 200 nm [Nayar et al. Biochim. Biophys.Acta. 986:200-206 (1989)]. Peptide-containing liposomes were thenseparated from non-encapsulated peptide by dialysis and finallysterilized by filtration through sterile membranes of 0.22 micrometers.A lipid composition of distearoyl-phosphatidylcholie, cholesterol anddistearoyl-phosphatidylethanlamine-polyethylene glycol (2,000), at amolar ratio of 5:4:0.3, was chosen. The amount of encapsulated peptidewas determined using the intrinsic fluorescence of Ang-(1-7).Encapsulation was achieved with an efficiency of 12% and a peptide/lipidratio of 0.03 (p/p). The size of liposomes was determined through thedynamic light scattering technique. A mean vesicle diameter of 0.19micrometer was determined.

Ang-(1-7)-containing liposomes (LAng) were unilaterally microinjected(35 ng of Ang-(1-7) in 200 nL) in the rostroventrolateral medulla (RVLM)of Wistar rats with a needle (30 G) that was inserted slowly in thebrain. Empty liposomes (LEmp) were also similarly microinjected at thesame lipid dose. The mean arterial blood pressure (MAP) was determinedby telemetry 4 days before and 12 days after microinjection in freelymoving undisturbed animals.

The microinjection of LAng produced a significant pressor effect duringday-time that was maintained for 5 days. The highest MAP was obtained onday 3 (114.+-0.4 mmHg) that differed significantly from that registeredon day 0 (100.+-0.3 mmHg). As expected, LEmp did not produce significantalteration of MAP (94.+-0.5 mmHg in 3 vs 90.+-0.5 mmHg in 0). Moreover,day-time MAP was significantly higher in LAng group than in

Lemp group on day 1, 2 and 3. Night-time MAP, in contrast to day-timeMAP, was not affected significantly by the microinjection of LAng.

Previous studies established that microinjection of free Ang-(1-7) (notencapsulated) in the RVLM, at a similar dose (25-50 ng), produced a 15mmHg increase of

PAM for approximately 10 min The short duration of this effect wasattributed to the elevated metabolism of the peptide in the free form.Therefore, the present technology established, in chronic conditions,the pressor effect of Ang-(1-7) at the level of RVLM. It is alsocharacterized by the capacity to increase the bioavailability of thepeptide.

EXAMPLE 2

Preparation of the microspheres in the basis of biodegradable polymer(PLGA) of Ang-(1-7) for the controlled release of the peptide.

Firstly a emulsion constituted of an organic phase constituted ofpoly(acid lactic-glycolic) (PLGA) dissolved in dichloromethane and anaqueous phase constituted of the 1.8 mg of Ang-(1-7). That emulsion isthen submitted to the sonication for half minute and is added to 1%(PVA) solution, forming a second emulsion, which suffers stirring for 1minute to complete homogenization of the microemulsion. The system ismaintained under stirring without heating, for 2 hours until theevaporation of the solvent. The mixture is centrifuged by 2 to 3 times,and washed three times with water to remove the surface-adsorbed PVA andfinally resuspended in 2 mL of water and freeze-dried. Then the solidmicrospheres were characterized through the thermal analysis andscanning electron microscopy SEM. The microspheres DSC curve shown avitreous transition similar to which it was observed to the PLGApolymer. The respectively SEM micrographs shown 50 microns of particlessize. It is still verified the porous surface of the microspheres. Todetermine the peptide encapsulation was accomplished by radioimmunoassay[Neves et al., Biochem. Pharmacol. 50:1451-1459 (1995)]. It was obtained15% of peptide encapsulation. The kinetics profile shown the 60% ofpeptide release approximately in 8 h and about 90% in 48 h.

EXAMPLE 3

Preparation of the inclusion compounds between β-cyclodextrin andAng-(1-7).

The preparation is made in equimolar proportions of cyclodextrin andAng-(1-7). In briefly, β-cyclodextrin and/or its derivatives isdissolved in water using stirring and heating. Then the respectiveamount of angiotensin-(1-7) is added to the aqueous solution. Followingthe dissolution, the mixture is frozen in liquid nitrogen and submittedtc the lyophilization process, obtaining a dry solid. The solid obtainedis then submitted to the physical-chemistry characterization using theFT infrared spectroscopy, thermal analysis (TG/DTG and DSC), X-raydiffraction and 1 H and 13 C NMR spectroscopy and T1 relaxation times.

EXAMPLE 4

This example describes the identification of an interaction betweenangiotensin-(1-7) and its analogues with the G protein-coupled receptor,MAS.

Angiotensin-(1-7) labeled with 125I or rhodamine-angiotensin-(1-7),fluorescent, were incubated with mouse kidney slices from normal or MASknockout animals. After incubation for variable intervals at 4oC theslices were exposed to autoradioghraphic films or analysed byflourescent microscopy. In the knockout mice the specif binding forangiotensin-(1-7) disappeared while the binding for Ang II or Ang IV,used as controls, was inaltered. The Ang_(1-7) binding in kidney slicesof wild type mice was displaced by the analoguesD-Ala⁷-Angiotensin-(1-7) e D-Pro⁷-Angiotensin-(1-7). The functional testfor the absence of binding in knockout mice was made using the waterdiuresis model (administration of 5% of the body weight, of H₂O).Ang-(1-7) treatment (4 pmol/10 g BW) in wild type mice produced areduction of the urine volume (antidiuresis). In MAS knockout mice theantidiuretic effect of Ang-(1-7) was absent.

1. A pharmaceutical formulation comprising Angiotensin-(1-7) or ananalogue or derivative thereof encapsulated in a liposome.
 2. Thepharmaceutical formulation of claim 1, wherein the Angiotensin-(1-7),analogue or derivative thereof is complexed with a hydrophiliccyclodextrin.
 3. The pharmaceutical formulation of claim 1, wherein theanalogue or derivative of Angiotensin-(1-7) is a member selected fromthe group comprising Sar¹-Angiotensin-(1-7), D-Ala⁷-Ang-(1-7) andD-Pro⁷-Ang-(1-7).
 4. The pharmaceutical formulation of claim 1, whereinthe liposome comprises one or more lipids selected from the groupcomprising phosphatidylcholine, phosphatidylserine,phosphatidylglycerol, cardiolipin, cholesterol, phosphatidic acid,sphingolipids, glycolipids, fatty acids, sterols,phosphatidylethanolamine, polymerizable lipids in their polymerized ornon-polymerized form, and combinations thereof.
 5. The pharmaceuticalformulation of claim 1, wherein the liposome is sterically-stabilizedwith a polyethylene glycol-lipid.
 6. The pharmaceutical formulation ofclaim 1, wherein the diameter of the liposome is below about 200 nm. 7.The pharmaceutical formulation of claim 2, wherein the hydrophiliccyclodextrin is an alpha-, beta- or gamma-cyclodextrin.
 8. Thepharmaceutical formulation of claim 2, wherein the hydrophiliccyclodextrin is a member selected from the group comprising6-O-maltosyl-,-cyclodextrin, sulfobutyl-,-cyclodextrin, 2-hydroxyethylcyclodextrin, 2-hydroxypropyl cyclodextrin, 3-hydroxypropylcyclodextrin, and 2,3- dihydroxypropyl cyclodextrin.
 9. A pharmaceuticalformulation, comprising Angiotensin-(1-7) or an analogue or derivativethereof encapsulated in synthetic biodegradable polymer or derivativethereof
 10. The pharmaceutical formulation of claim 9, wherein theAngiotensin-(1-7) or an analogue or derivative thereof is complexed witha hydrophilic cyclodextrin.
 11. The pharmaceutical formulation of claim9, wherein the polymer is a member selected from the group comprisingpolyanhydrides, poly(hydroxy acids), poly(hydroxyl)acids, polyuretans,polysyloxana, polymethacrylates, polyvinyl alcohol,poly(2-hydroxy-ethylmethacrylate), polyacrylamide, lactic acid polymers(PLA), glycolic acid polymers (PGA), and co-polymers thereof.
 12. Thepharmaceutical formulation of claim 10, wherein the hydrophiliccyclodextrin is an alpha-, beta- or gamma-cyclodextrin.
 13. Thepharmaceutical formulation of claim 10, wherein the hydrophiliccyclodextrin is a member selected from the group comprising6-O-maltosyl-,-cyclodextrin, sulfobutyl-,-cyclodextrin, 2-hydroxyethylcyclodextrin, 2-hydroxypropyl cyclodextrin, 3-hydroxypropylcyclodextrin, and 2,3- dihydroxypropyl cyclodextrin.
 14. Thepharmaceutical formulation of claim 9, wherein the analogue orderivative of Angiotensin-(1-7) is a member selected from the groupcomprising Sar¹-Angiotensin-(1-7), D-Ala⁷-Ang-(1-7) andD-Pro⁷-Ang-(1-7).
 15. A medical device for controlled-release ofAngiotensin-(1-7), an analogue or a derivative thereof, comprisingAngiotensin-(1-7), the analogue or the derivative thereof encapsulatedin a synthetic biodegradable polymer or derivative thereof.
 16. Amedical device for controlled-release of Angiotensin-(1-7), an analogueor a derivative thereof, comprising Angiotensin-(1-7), the analogue orthe derivative thereof complexed with a hydrophilic cyclodextrin. 17.The device of claim 15, wherein the synthetic biodegradable polymer isselected from the group comprising polyanhydrides, poly(hydroxy acids),poly(hydroxyl)acids, polyuretans, polysyloxana, polymethacrylates,polyvinilalcholy, poly(2-hydroxy-ethylmethacrylate), polyacrylamide,lactic acid polymers (PLA), glycolic acid polymers (PGA), andco-polymers thereof.
 18. The device of claim 16, wherein the hydrophiliccyclodextrin is an alpha-, beta- or gamma-cyclodextrin.
 19. The deviceof claim 16, wherein the hydrophilic cyclodextrin is a member selectedfrom the group comprising 6-O-maltosyl-,-cyclodextrin,sulfobutyl-,-cyclodextrin, 2-hydroxyethyl cyclodextrin, 2-hydroxypropylcyclodextrin, 3-hydroxypropyl cyclodextrin, and 2,3-dihydroxypropylcyclodextrin.
 20. The device of claim 15, wherein the analogue orderivative of Angiotensin-(1-7) is a member selected from the groupcomprising Sar¹-Angiotensin-(1-7), D-Ala⁷-Ang-(1-7) andD-Pro⁷-Ang-(1-7).
 21. A method for treating or preventing acardiovascular disease or cancer in an animal, comprising administeringto the animal a therapeutically effective amount of Angiotensin-(1-7) oran analogue or derivative thereof.
 22. The method of claim 21, whereinthe Angiotensin-(1-7) or an analogue or derivative thereof is complexedwith a hydrophilic cyclodextrin.
 23. The method of claim 22, wherein thehydrophilic cyclodextrin is an alpha-, beta- or gamma-cyclodextrin. 24.The method of claim 22, wherein the hydrophilic cyclodextrin is a memberselected from the group comprising 6-O-maltosyl-,-cyclodextrin,sulfobutyl-,-cyclodextrin, 2-hydroxyethyl cyclodextrin, 2-hydroxypropylcyclodextrin, 3-hydroxypropyl cyclodextrin, and 2,3-dihydroxypropylcyclodextrin.
 25. The method of claim 21, wherein the Angiotensin-(1-7)or the analogue or derivative thereof is encapsulated in a syntheticbiodegradable polymer or derivative thereof.
 26. The method of claim 25,wherein the synthetic biodegradable polymer or derivative thereof is amember selected from the group comprising polyanhydrides, poly(hydroxyacids), poly(hydroxyl)acids, polyuretans, polysyloxana,polymethacrylates, polyvinyl alcohol, poly(2-hidroxi-ethylmetacrilate),polyacrylamide, lactic acid polymers (PLA), glycolic acid polymers(PGA), and co-polymers thereof.
 27. The method of claim 21, wherein theAngiotensin-(1-7) or the analogue or derivative thereof is encapsulatedin a liposome.
 28. The method of claim 27, wherein the liposomecomprises one or more lipids selected from the group comprisingphosphatidylcholine, phosphatidylserine, phosphatidylglycerol,cardiolipin, cholesterol, phosphatidic acid, sphingolipids, glycolipids,fatty acids, sterols, phosphatidylethanolamine, polymerizable lipids intheir polymerized or non-polymerized form, and combinations thereof. 29.The method of claim 21, wherein the analogue or derivative ofAngiotensin-(1-7) is a member selected from the group comprisingSar¹-Angiotensin-(1-7), D-Ala⁷-Ang-(1-7) and D-Pro⁷-Ang-(1-7).
 30. Themethod of claim 21, wherein the cardiovascular disorder is selected fromthe group comprising left ventricular hypertrophy, myocardial ischemia,stroke, heart failure, atherosclerosis, coronary heart disease,myocardial infarction (not specifically enumerated), angina pectoris,endothelial dysfunction.
 31. A method for treating or preventing adisease in an animal caused by a reduced function or stimulation of theG-protein-coupled receptor MAS, comprising administering atherapeutically effective amount of a pharmaceutical formulationcomprising Angiotensin-(1-7) or an analogue or derivative thereofencapsulated in liposomes or bound to a biodegradable polymer.
 32. Thedevice of claim 16, wherein the analogue or derivative ofAngiotensin-(1-7) is a member selected from the group comprisingSar¹-Angiotensin-(1-7), D-Ala⁷-Ang-(1-7) and D-Pro⁷-Ang-(1-7).